This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Imaging oxygen in 3D with sub-micron spatial resolution can be made possible by combining phosphorescence quenching technique with multiphoton laser scanning microscopy. However, Pt and Pd porphyrin-based phosphorescent dyes, traditionally used as phosphors in biological oxygen sensing, exhibit extremely low two-photon absorption (2PA) cross-sections. Using chromophors with large known two photon cross sections linked to porphyrins, we are investigating the possibility of generating high phosphorescence yields after photoexcitation and subsequent energy transfer between chormophor and porphyrin. Such complexes would be highly desirable for single molecule (SM) studies employing phosphorescence instead of fluorescence. Up to this point, the use of phosphorescence is quite limited in SM techniques due to the low level of signal. Oxygen dependent quenching of phosphorescence is a sensitive method for measuring molecular oxygen. Our proposed complexes could play a key role in measuring molecular oxygen in tissue employing an imaging technique in vivo. Specific aims of our project include: (1) Synthesize 2P absorbing phosphorescent probes, in which 2P antenna dyes are separated from the triplet emitter by a dendritic "insulator," to prevent intamolecular quenching of the phosphorescence via electron transfer. (2) Modify the existing confocal microscope system by coupling it with a femtosecond Ti:Sapphire laser and a time domain phosphorometer to perform 2P excitation of antenna chromophores, scanning the object in axial planes under the surface and measuring phosphorescence lifetimes. (3) Align and test the entire setup using an appropriate model with microscopic oxygen gradients. Validate the technique by performing in vivo oxygen imaging in the dorsal window tumor model in rats.